PolyI:C Upregulated CCR5 and Promoted THP-1-Derived Macrophage Chemotaxis via TLR3/JMJD1A Signalling

Objective This study aimed to evaluate the specific roles of polyinosinic:polycytidylic acid (polyI:C) in macrophage chemotaxis and reveal the potential regulatory mechanisms related to chemokine receptor 5 (CCR5). Materials and Methods In this experimental study, THP-1-derived macrophages (THP1-Mφs) induced from THP- 1 monocytes were treated with 25 μg/mL polyI:C. Toll-like receptor 3 (TLR3), Jumonji domain-containing protein (JMJD)1A, and JMJD1C small interfering RNA (siRNAs) were transfected into THP1-Mφs. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) was used to detect the expression levels of TLR3, CCR5, 23 Jumonji C domain-containing histone demethylase family members, JMJD1A, and JMJD1C in THP1-Mφs with different siRNAs transfections. Western blot was performed to detect JMJD1A, JMJD1C, H3K9me2, and H3K9me3 expressions. A transwell migration assay was conducted to detect THP1-Mφ chemotaxis toward chemokine ligand 3 (CCL3). A chromatin immunoprecipitation (ChIP) assay was performed to detect H3K9me2-CCR5 complexes in THP1- Mφs. Results PolyI:C significantly upregulated CCR5 in THP1-Mφs and promoted chemotaxis toward CCL3 (P<0.05); these effects were significantly inhibited by TLR3 siRNA (P<0.01). JMJD1A and JMJD1C expression was significantly upregulated in polyI:C-stimulated THP1-Mφs, while only JMJD1A siRNA decreased CCR5 expression (P<0.05). JMJD1A siRNA significantly increased H3K9me2 expression in THP1-Mφs but not in polyI:C-stimulated THP1-Mφs. The ChIP result revealed that polyI:C significantly downregulated H3K9me2 in the promoter region of CCR5 in THP1- Mφs. Conclusion PolyI:C can enhance THP1-Mφ chemotaxis toward CCL3 regulated by TLR3/JMJD1A signalling and activate CCR5 expression by reducing H3K9me2 in the promoter region of CCR5.


Introduction
Acute lung injury (ALI) is an inflammation characterized by the breakdown of the endothelial and epithelial lung barrier (1). Monocyte-derived macrophages are important in the pathogenesis of ALI. Under the pathological conditions of ALI, activated circulating monocytes infiltrate the alveolar space to form alveolar macrophages. Subsequently, alveolar macrophages may secrete several inflammatory mediators, such as cytokines and chemokines, to induce the migration of mature neutrophils and CD4 + T cells into the alveolar space, thereby prompting an inflammation response that may kill pathogenic microbes (2,3). A previous study showed that the depletion of circulating monocytes and subsequently recruited alveolar macrophages significantly suppressed ALI in mice (4). Therefore, the function and activity of macrophages are extremely important in the development and prognosis of ALI.
Toll-like receptors (TLRs) are categorized as innate immune sensors, which play an important role in the process of antigen recognition for innate immune cells such as macrophages (5). It has been reported that TLR3 is upregulated in alveolar macrophages throughout the ALI pathogenesis (6). Chemokines comprise a class of cytokines that act as signalling molecules in the regulation of inflammatory response (7). Chemokine receptors (CCRs) are specific receptors for chemokines that are integral to the recruitment of alveolar macrophages (8). TLR3 and CCRs participate in ALI-induced inflammatory response through the recognition of pathogen-related molecular processes or the recruitment of macrophages; however, whether a direct regulating mechanism between CCRs and TLR3 exists in macrophages has not been thoroughly researched.
Histone demethylation is an important form of epigenetic modification that is regulated by Jumonji C domaincontaining histone demethylases (JHDMs) (9). Histone demethylation is involved in the transcriptional repression and activation of target genes, and is closely associated with the inflammatory response of macrophages. It has been reported that Jumonji domain-containing protein 3 (JMJD3) influences transcriptional gene expression in lipopolysaccharide (LPS)-activated macrophages, and the regulatory role of JMJD3 is dependent upon H3K4me3 Regulatory Effects of polyI:C on Macrophages (10). An H3K27me3 inhibitor reduces LPS-induced proinflammatory cytokine production by macrophages, and this process is regulated by UTX and JMJD3 (11). Moreover, a pervious study reported that high glucose upregulates diverse inflammatory cytokines in macrophages, including IL-6, IL-12p40, and MIP-1α/β; this process is closely associated with H3K9 methylation (12). However, the specific role of H3K9 methylation in TLR3 signalling for macrophage-involved inflammatory responses remains unknown.
Polyinosinic:polycytidylic acid (PolyI:C) is a viral mimetic that mimics inflammatory responses to systemic viral infection (13). In this study, the effects of polyI:C on THP-1-derived macrophage (THP1-Mφ) chemotaxis, as well as potential regulatory mechanisms related to TLR3 and CCRs, are explored. The aim of this study is to provide new insight into the underlying regulatory mechanisms for macrophage participation in ALI.

Cell culture and induction of THP-1-derived macrophages (THP1-Mφs)
In this experimental study, human THP-1 monocytes were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 medium that contained 10% heat-inactivated foetal bovine serum (FBS, Gibco, USA) and 100 U/ mL penicillin-streptomycin. Cells were maintained in an atmosphere of 5% CO 2 at 37˚C. Exponential-phase cells were used in the following assays.

Quantitative real-time reverse transcriptase polymerase chain reaction
Total RNA was extracted from cells of different groups using TRIzol (Fermentas, Burlington, Ontario, Canada) and reverse-transcribed by RevertAid M-MuLV Reverse Transcriptase (Fermentas, Canada) in accordance with the manufacturer's instructions. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) was performed on a LightCycler 2.0 Instrument (Roche, Germany) using the SYBR Green PCR Kit (TaKaRa, Japan). The relative expression levels of target genes were calculated by 2 -ΔΔCt , using GAPDH as an internal control. The primer sequences are shown in Table 1.

Flow cytometry
Flow cytometry was performed to detect chemokine receptor 5 (CCR5) expression in THP1-Mφs. Simply, cells were suspended in fresh RPMI-1640 medium and incubated with CCR5-PE antibody (R&D Systems, USA) in the dark for 30 minutes at room temperature. Data were collected using the FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analysed with CellQuest software (BD Biosciences).
After 6 hours of incubation with 100 ng/mL PMA, THP1-Mφs were incubated with specific siRNAs and Lipofectamine 2000 reagent (ThermoFisher, Waltham, MA, USA) for 6 hours. Transfected cells were treated with 25 μg/ mL polyI:C for an additional 42 hours. The efficacy of the TLR3 transfection was detected using qRT-PCR and flow cytometry as described above, while the efficacy of JMJD1A and JMJD1C siRNA-mediated gene silencing was monitored using Western blotting.

Transwell migration assay
THP1-Mφ chemotaxis toward chemokine ligand 3 (CCL3) was detected using transwell inserts. Transwell inserts with a pore size of 8 μm were placed into 24-well plates. Cells were suspended in serum-free RPMI-1640 medium and inoculated into the upper chamber at a density of 1×10 5 cells/mL. RPMI-1640 medium that contained 100 ng/mL recombinant human CC chemokine ligand 3 (rhCCL3;#270-LD, R&D Systems, USA) and 10% FBS was added into the lower chamber. Following 12 hours of incubation at 37˚C, the non-migrated cells were removed from the upper chamber, and migrated cells in the lower chamber were fixed with methanol and stained with eosin. Five random fields of each well were observed using light microscopy, and the number of migrated cells was counted.

Chromatin immunoprecipitation assay
The chromatin immunoprecipitation (ChIP) assay was performed to detect H3K9 methylation in THP1-Mφs. After being fixed in 1% formaldehyde, the chromatin was extracted from THP1-Mφs using sonication. Then, the chromatin was immunoprecipitated with H3K9me2 (Abcam, Cambridge, MA, USA) or H3K9me3 antibody (Abcam, USA) pre-bound Protein G-plus Agarose beads, overnight at 4˚C. Precipitated protein-DNA complexes were eluted in Tris-EDTA buffer that contained 2% sodium dodecyl sulfonate (SDS), and the crosslink was reversed through a 16 hour incubation period at 65˚C. The precipitated DNA fragments were analysed by qRT-PCR as described above. The primer sequences of CCR5-ChIP are shown in Table 1. qRT-PCR was performed on a LightCycler 2.0 Instrument (Roche, Germany) using TB Green Fast qPCR Mix (Code No. RR430S/A/B, TaKaRa, Japan).

Western blot
THP1-Mφs were lysed in RIPA buffer. Total proteins were separated by SDS-polyacrylamide gel electrophoresis on 10% polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% skim milk in TBST for 2 hours and incubated with special primary antibody (anti-H3K9me2, anti-H3K9me3, Abcam, USA) at 4˚C for 12 hours. After there were washed three times with TBST, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA) at 25˚C for 2 hours. Protein bands were visualized with the Image Station IS2000 (Kodak, Rochester, NY, USA).

Statistical analysis
All experiments were performed in triplicate, and all data are presented as means ± standard deviation. The statistical analysis conducted in this study was performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). The Shapiro-Wilk was used to test the normality of the distribution. For the data presenting a normal distribution, the mann-withney (two groups) and kruskal-wallis (more than two groups) were used to compare results among different groups. The Wilcoxon rank-sum test was used for non-normally distributed data. P<0.05 denoted statistically significant results.
Since H3K9 is known to be the substrate of JMJD1A, we sought to determine if the regulatory role of JMJD1A in CCR5 expression was dependent on H3K9 methylation. As shown in Figure 4D, H3K9me2 expression was decreased in polyI:C-treated THP1-Mφs, while H3K9me3 expression was not significantly changed. In addition, H3K9me2 was significantly upregulated by JMJD1A siRNA transfection in THP1-Mφs. However, H3K9me3 expression was not influenced by JMJD1A siRNA transfection in polyI:Cstimulated THP1-Mφs (Fig.4E). In addition, polyI:C treatment downregulated H3K9me2 expression in the promoter region of CCR5 in THP1-Mφs (Fig.4F).

Discussion
Macrophage chemotaxis is an important component of ALI pathogenesis. It is known that viral infections can induce alveolar macrophage recruitment, but the regulatory mechanisms of viral infection (polyI:C) on monocyte-derived macrophages are still unclear. Thus, in this study, we have explored the regulatory mechanisms of polyI:C on THP1-Mφs. The results showed that polyI:C significantly upregulated CCR5 in THP1-Mφs and promoted THP1-Mφ chemotaxis toward CCL3 via TLR3 signalling. In addition, polyI:C-upregulated CCR5 was mediated by JMJD1A, and H3K9me2 was downregulated in the promoter region of CCR5 in THP1-Mφs.
Since CCRs are important in macrophage chemotaxis, the expression levels of diverse CCRs were examined in THP1-Mφs after polyI:C treatment. Our results demonstrated that only CCR5 was significantly upregulated by polyI:C treatment in THP1-Mφs. CCR5 is a cell surface G proteincoupled receptor that is involved in inflammatory response via interaction with specific chemokine ligands, including CCL3, CCL4, and CCL5 (14)(15)(16). The activation of CCR5 and CCL5 is required to prevent the apoptosis of virusinfected macrophages (17). In addition, CCR5 is involved in obesity-induced adipose tissue inflammation via regulation of macrophage recruitment (18,19). Moreover, it has been reported that polyI:C-treated macrophages can promote CCR5 expression (20), which is consistent with the findings of our study. It was supposed that CCR5 is involved in polyI:C-induced inflammation in THP1-Mφs. Subsequently, THP1-Mφ chemotaxis toward CCL3 (a ligand of CCR5) was investigated. The results suggest that polyI:C significantly increased THP1-Mφ chemotaxis toward CCL3. A previous study reported that CCL3 expression was significantly elevated in the lung of a murine model of LPS-induced ALI and mediated an enhanced inflammatory injury-possibly by recruiting macrophages (21). Therefore, polyI:C-upregulated CCR5 contributes to the promotion of macrophage chemotaxis by interacting with CCL3.
Moreover, our results also suggest that TLR3 siRNA transfection significantly suppressed CCR5 expression in polyI:C-stimulated THP1-Mφs and inhibited chemotaxis toward CCL3. TLR-3 is responsible for anti-viral immunity against several virus infections via double-stranded RNA recognition and the activation of multiple antiviral factors in macrophages (20). Similarly, TLR-3 is activated in macrophages in response to encephalomyocarditis infection via type 1 IFN production. It has been reported that CCR5 may participate in virus replication and acts as the primary receptor for regulating encephalomyocarditis infection in mediating inflammatory response-related genes in macrophages (22). These results indicate that macrophages may recognize polyI:C stimulation through TLR3 signalling. PolyI:C may upregulate CCR5 expression and promote THP1-Mφ chemotaxis toward CCL3 through TLR3 signalling.
Histone demethylation, dynamically regulated by JHDMs, is implicated in the regulation of inflammatory response of macrophages (23). Previous studies have reported that JMJD3 is over-expressed in LPS-activated macrophages, which regulates diverse genes involved in LPS-induced immune and inflammatory responses (10,24). However, few studies have focused on the regulatory mechanisms of polyI:C in histone demethylation in macrophages. In this study, the expression levels of 23 JHDM family members were detected in polyI:Cstimulated THP1-Mφs. The expression levels of JMJD1A, JMJD1C, JMJD2A, JARID1A, and HSPBAP1 were significantly increased by polyI:C in THP1-Mφs, while that of JMJD3 was not significantly changed. These results indicated that the effects of polyI:C on inflammatory responses of macrophages might differ from LPS. Since JMJD1A and JMJD1C could be regulated by TLR3 in polyI:C-stimulated THP1-Mφs, the regulatory roles of JMJD1A and JMJD1C on CCR5 were further analysed in this study. It was revealed that CCR5 was significantly downregulated by JMJD1A siRNA transfection in polyI:Cstimulated THP1-Mφs, while CCR5 expression was not significantly influenced by JMJD1C siRNA transfection. The regulatory role of JMJD1A has been found to affect the proliferation, migration, and invasion of cancer cells in various cancer types (25)(26)(27). It has been reported that JMJD1A inhibition suppresses tumour growth by downregulating angiogenesis and macrophage infiltration (28). Our findings indicate that polyI:C treatment may induce a similar macrophage inflammatory response with cancer; PolyI:C may enhance CCR5 expression by upregulating JMJD1A in THP1-Mφs.
Since JMJD1A is a H3K9 demethylase, the H3K9 methylation state of CCR5 was analysed in polyI:Cstimulated THP1-Mφs. Our results showed that H3K9me2 expression was significantly decreased by polyI:C treatment in THP1-Mφs. H3K9me2 downregulation might have attributed to the upregulation of JMJD1A. However, H3K9me3 expression was not significantly influenced by polyI:C treatment. Our findings indicate that the regulatory role of JMJD1A on CCR5 was dependent on H3K9me2. In addition, H3K9me2 was upregulated by JMJD1A siRNA transfection in THP1-Mφs, while H3K9me2 expression was not significantly influenced by JMJD1A siRNA in polyI:C-stimulated THP1-Mφs. This may be explained by the fact that some other upregulated JHDMs induced by polyI:C, such as JMJD1C, and JMJD2A may share a target with JMJD1A. JMJD1C and JMJD2A exhibit redundant effects on H3K9me2 expression. The presence of H3K9me2 in the promoter region of target genes typically results in reduced expressions of its targets. A previous study has reported that H3K9 exhibits a low methylation level in response to the activation of dendritic cells and is erased from the promoters of some activated inflammatory genes (29). Consistent with the results of that study, our results reveal that H3K9me2 expression was significantly reduced by polyI:C treatment in the promoter region of CCR5 in THP1-Mφs. We suspected that polyI:C-mediated JMJD1A upregulation may upregulate CCR5 by reducing H3K9me2 in the promoter region of CCR5. Interestingly, JMJD1A is also a hypoxiainducible gene that has been found to be upregulated in hypoxia-stimulated macrophages. However, hypoxia treatment decreases CCR5 expression via H3K9me2 upregulation in the promoter region of CCR5 (30). This may be explained by the effects of hypoxia-induced repressive JMJDs, which can overwhelm the effects of JMJD1A.

Conclusion
The present study revealed that polyI:C upregulated JMJD1A expression in THP1-Mφs, thereby elevating the CCR5 expression by reducing H3K9me2 in the promoter region of CCR5 via TLR3 signalling. However, this study is still limited to the cellular level, and the validation of these results in animal models is required in future research.